869 869 869 869 869 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(8): 869-873, D ecem ber 2005
A genotyping study of human immunodeficiency virus type-1 drug
resistance in a small Brazilian municipality
Walter A Eyer-Silva/* /* * /+, M ariza G M orgado
Laboratório de Aids & Imunologia Molecular, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil *Hospital Universitário Gaffrée e Guinle, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
**Programa Municipal de HIV/Aids de Miracema, Miracema, RJ, Brasil
In Brazil, surveillance studies on antiretroviral drug resistance among drug-naïve and treatment-experienced patients have focused primarily on patients living in large urban centers. As the epidemic spreads towards small municipalities and the innermost parts of the country, it will be essential to monitor the prevalence of antiretroviral drug resistance in these areas. We report the first survey on the prevalence of antiretroviral drug resistance in a small Brazilian municipality. Between July 1999 and March 2005, 72 adult human immunodeficiency virus type-1(HIV-1)-infected patients received care at the Municipal HIV/AIDS Program of the small, southeastern mu-nicipality of Miracema, state of Rio de Janeiro. A genotyping study of antiretroviral drug resistance was performed in 54 patients. Among 27 samples from treatment-experienced patients, 9 (33.3%) harbored strains with reduced drug susceptibility. Among these, 6 had reduced susceptibility to reverse transcriptase (RT) inhibitors and 3 to both RT and protease inhibitors. No primary antiretroviral drug resistance was recorded among 27 drug-naïve subjects. The relatively low prevalence of resistance mutations in the Miracema cohort argues against the concern that resource-poor settings should not implement widespread accessibility to standard of care antiretroviral combina-tions due to the possibility of sub-optimal adherence leading to the emergence and spread of drug-resistant strains.
Key words: antiretroviral resistance genotyping human immunodeficiency virus type1 small municipalities subtypes -Brazil
Continued human immunodeficiency virus type 1 (HIV-1) replication in the presence of selective pressure of drugs targeting the reverse transcriptase (RT) and protease (PR) viral enzymes leads to the emergence of specific point mutations in the RT and PR genomic regions of the poly-merase (pol) gene and poses one of the major obstacles to the long-term efficacy of antiretroviral therapy. Since strains harboring resistance-associated mutations to a single or to multiple antiretroviral agents can be transmit-ted both horizontally (Hecht et al. 1998) and vertically (Siegrist et al. 1994), it is essential to monitor the preva-lence of resistant strains in the community.
In Brazil, surveillance studies on the prevalence of mutations conferring antiretroviral drug resistance are being reported both from national (Brindeiro et al. 1999, 2003, Tanuri et al. 2002) and regional (Dumans et al. 2002, Pires et al. 2004, Couto-Fernandez et al. 2005, Rodrigues et al. 2005) studies based on large urban centers. How-ever, as the Brazilian epidemic spreads from the large ur-ban centers towards small municipalities and the inner-most parts of the country (Szwarcwald et al. 2000), appro-priate studies on the features of HIV-1 infection in rela-tively small Brazilian communities are urgently needed. We report the results of the first survey on antiretroviral drug resistance in a small Brazilian municipality: the south-eastern Miracema, in the state of Rio de Janeiro. Since Miracema is located in northwestern Rio de Janeiro, a
re-+Corresponding author. E-mail: walter-eyer@unirio.br
Received 31 May 2005 Accepted 23 November 2005
gion where municipalities are known to have low human development index values (IPEA 2003), the present study is an opportunity to analyze the prevalence of HIV-1 drug resistance in a resource-poor setting with widespread availability of standard of care highly active antiretroviral therapy (HAART) regimens.
MATERIAL AND METHODS
Patients and setting - Miracema is a small municipal-ity in northwestern state of Rio de Janeiro (21°24’50”S; 42°11’52”W), 280 km far from the city of Rio de Janeiro, at the border of the state of Minas Gerais. A detailed clinical and epidemiological characterization of the cohort is pre-sented elsewhere (Eyer-Silva et al. 2005). Monthly medi-cal appointments are offered at a lomedi-cal ambulatory facility with a physician based in the city of Rio de Janeiro. Anti-retroviral agents are freely supplied to patients when clini-cally indicated, as part of the national AIDS Program of the Ministry of Health. Patients were staged according to the 1993 Revised Classification System of the Centers for Disease Control and Prevention (CDC 1992). The study protocol was reviewed and approved by the Ethics Re-view Board at Instituto de Pesquisas Clínicas Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro.
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870 HIV-1 genotyping in a small Brazilian municipality• WA Eyer-Silva, M G M orgado
was amplified in the following mixture: 5 µl of extracted DNA, 5 µl of 10 ×Taq buffer, 0.6 µl of 25mM (each) deoxy-nucleoside triphosphates, 5 µl of MgCl2 25mM, 1 µl (25 pM) of each PCR-primer, 33 µl of H20, and 0.5 µl of Taq
DNA polymerase (Amersham Pharmacia Biotech Inc.). An initial cycle was performed with a denaturation tempera-ture set at 95°C (3 min), annealing set at 55°C (1 min) and extension set at 72°C (1 min), followed by 35 cycles with denaturation at 95°C (1 min), annealing at 55° (45 s) and extension at 72°C (1 min). A final extension of 10 min was set at 72°C. For second-round PCR, a 5 µl aliquot of the first-round PCR mixture was used. Oligonucleotides DP10 (5'-TAACTCCCTCTCAGAAGCAGGAGCCG-3') and RT12 (5'-ATCAGGATGGAGTTCATAACCCATCCA-3') were used as sense and antisense outer primers, respectively. Oligonucleotides DP16 (5'-CCTCAAATCACTCTTT GGCAAC-3') and RT4 (5'-AGTTCATAACCCATCC AAAG-3') were used as sense and antisense nested prim-ers, respectively. Amplification results were checked on agarose gel electrophoresis and ethidium bromide stain-ing. The PCR products were then purified with the com-mercial Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, US) and sequenced in an auto-mated ABI 310 or 3100 autoauto-mated sequencer (Applied Biosystems, Foster City, CA, US) using primers DP16, RT4, LR49 CAATGG CCATTGACAGAAGA-3') and L51 (5'-TGTGGTAT CCTAATTGAACTTCCC-3') for pol frag-ments. Sequencing reaction mixtures were assembled with BigDye Terminator v.3.0 Cycle Sequencing Reaction Kits (Applied Biosystems). Chromatogram sequencing files were inspected with Chromas 1.45 (Technelysium Ltd., Queensland, Australia) and contigs were assembled by using SeqMan II, included in the DNASTAR software package (Promega) (Burland 2000). Sequences were de-posited on the GenBank database under the accession numbers AY929012 to AY929061 and DQ058780 to DQ058783.
Drug resistance genotyping - Previously described drug-resistance associated mutations in the PR and RT genes were sought and genotyping results were inter-preted by using the drug resistance interpretation beta test from the HIV RT and PR Sequence Database, Stanford University, version 4.1 (Kantor et al. 2001).
Analysis of sequences and phylogenetic studies - For subtyping analysis, sequences were aligned against a set of reference strains from all known HIV-1 group M sub-types (gathered from the Los Alamos HIV Database: http:/ /hiv-web.lanl.gov) and trimmed to equivalent lengths by using CLUSTAL X (Thompson et al. 1997). A SIVcpz se-quence was used as outgroup. An alignment of 845 bp (corresponding to positions 2364 to 3198 relative to HXB2 genome, GenBank accession no. K03455) was obtained. Phylogenetic inferences were performed by the neighbor-joining (NJ) algorithm (Saitou & Nei 1987) based on a DNA distance matrix and using the F84 model of nucle-otide substitution (Felsenstein 1984) implemented in PAUP* version 4.0b10 (Swofford 2002). The robustness of the trees was evaluated by bootstrap analysis (Fel-senstein 1985) with 100 rounds of replication. The bootscanning method was used to detect and study
re-combination, as implemented in the SIMPLOT software, version 2.5 (Salminen et al. 1995). The analysis was per-formed on a sliding window of 400 nucleotides of the query sequences moving by increments of 20 nucleotides along an alignment of the reference sequences.
RESULTS
Between July 1999 and January 2005 a total of 72 HIV-1 infected adult patients (37 female) received care at the Municipal HIV-1/AIDS Program. Out of the 58 patients from whom a blood sample was available, pol sequences were obtained from 54 (we failed to obtain pol sequences from 4 samples, even after analysis of a second blood sample). Fifty samples were assigned subtype B, whereas strains M02, M08, M31, and M36 were BF1 mosaic forms (the first 3 sharing common intersubtype breakpoints). These BF1 recombinants will be further described else-where. No other subtypes were found.
The Table presents the demographic, epidemiologi-cal, clinical and virological data of 54 studied patients. As of sample collection, 27 patients were treatment-naïve, 23 had already been exposed to HAART and 4 were not on therapy but had been previously offered vertical trans-mission prophylaxis (VTP) with a combination of zido-vudine, lamivudine and nevirapine. Among treatment-ex-perienced patients, 10 were on a combination of 2 nucleo-side RT inhibitors (NRTI) plus 1 non-nucleonucleo-side RT in-hibitor (NNRTI), 9 were on a regimen of 2 NRTI plus 1 PR inhibitor (PI), and 4 were on a 2 NRTI plus 1 PI plus 1 NNRTI combination. A total of 29 patients were on an AIDS-defining CDC stage category (27 on stage C and 2 on stage A3).
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TABLE
Demographic, epidemiological, clinical and virological data from 54 adult human immunodeficiency virus type-1-infected patients followed at the Municipal HIV-1/AIDS Program of Miracema
Patient Sex Age CDC Year of Time on
stage sample therapy Drugs used on treatment a RT b TR mutations RT b PR mutations Subtype
M02 F 21 A3 2001 3.5 yr DDI, AZT, 3TC, NFV, NVP S None S K63P, V77I BF1
M03 F 20 A1 2005 VTP c AZT, 3TC, NVP S None S L63P, V77I B
M05 F 47 C3 2001 4.5 yr AZT, DDI, SAQ, IND, RIT, 3TC, D4T, NFV R D67N, K70R, K219E R M36I, L63P, A71T, N88D B
M06 F 26 C2 2001 3 mo AZT, 3TC, NFV S None S M36I, L63P B
M07 F 28 A2 2004 7 yr DDI, AZT, 3TC, LOP R D67N, K219Q S L63P B
M08 M 24 C3 2001 4.5 yr AZT, IND, 3TC, D4T, NFV, NVP R Y181C, G190A S L10I, V77I BF1
M11 M 27 C3 2001 2 yr AZT, 3TC, NFV R M184V R K20I, M36I, L63P, A71T, N88S B
M12 M 53 A2 2002 5 yr AZT, SAQ, NFV, 3TC, D4T, LOP R T69S, K70R, M184V R M36I, L63T, A71T, N88D B M14 M 34 C3 2001 3 yr AZT, DDI, D4T, 3TC, EFV R M41L, L210W S L63T, V77I B
M16 F 35 A1 2001 VTP AZT, 3TC, NVP S None S L63P B
M17 M 31 C3 2001 5 yr AZT, DDI, 3TC, D4T, EFV S None S L63P B
M20 F 27 C3 2001 18 mo AZT, 3TC, EFV R M184V S K20T B
M21 F 26 A2 2001 naïve — S None S L63P B
M22 F 35 C3 2001 2.5 yr DDI, AZT, 3TC, NFV S None S L63P B
M23 F 42 A2 2001 naïve — S None S L63T B
M24 F 22 A1 2002 naïve — S None S None B
M25 F 24 A2 2003 6 yr DDI, AZT, 3TC, D4T, NVP R K103N S L63P B
M28 M 33 C3 2001 4 yr AZT, DDI, SAQ, NFV, 3TC, D4T, LOP, EFV S None S L63P B M31 F 60 B2 2003 5 yr AZT, DDI, RIT, D4T, 3TC, NFV, EFV R M184V S L63P, V77I BF1 M33 F 27 A1 2001 VTP AZT, 3TC, NVP S V106I, P225L S M36I, L63P B
M34 F 16 A1 2004 VTP AZT, 3TC, NVP S None S L63P B
M36 F 38 A3 2002 1 yr AZT, 3TC, EFV S None S M36I, D60E, V77I BF1
M37 M 30 A1 2002 naïve — S None S L63P B
M38 F 57 B2 2002 naïve — S None S L63P B
M39 F 30 C3 2001 naïve — S None S None B
M40 F 24 A2 2001 naïve — S None S None B
M41 F 26 C3 2001 naïve — S None S L63P, I93L B
M42 M 32 C3 2001 2 mo AZT, 3TC, EFV S V118L S M36I, L63P B
M43 M 24 A1 2002 naïve — S None S M36I, L63P B
M44 F 33 A1 2002 naïve — S None S M36I, L63P B
M45 F 22 A1 2002 naïve — S None S L63P, A71T B
M46 M 37 C3 2002 6 mo AZT, 3TC, EFV S None S L33F, L63T, V77I B
M47 M 29 C2 2002 naïve — S None S L63P, V77I, I93L B
M48 M 41 C3 2002 3 mo AZT, D4T, 3TC, EFV S None S L63P, A71T B
M49 M 30 C3 2002 3 mo AZT, 3TC, EFV S None S L63T, V77I B
M50 F 20 B2 2002 naïve — S None S None B
M51 M 23 A2 2002 naïve — S None S None B
M52 M 25 C3 2002 naïve — S None S None B
M53 M 31 C3 2002 naïve — S None S L63P, I93L B
872 872 872 872
872 HIV-1 genotyping in a small Brazilian municipality• WA Eyer-Silva, M G M orgado
DISCUSSION
Little is known about the features of HIV-1 infection in small Brazilian municipalities and rural areas. As the epi-demic spreads from the large urban centers towards small municipalities and the innermost parts of the country (Szwarcwald et al. 2000, Brito et al. 2001), there is an ur-gent need to study the clinical, epidemiological and viro-logical characteristics of HIV-1 infection in these settings. These studies will be of utmost importance to optimize the institution of adequate control measures, grasp the true magnitude of the problem, improve clinical recogni-tion and management of HIV-1 infecrecogni-tion and better allo-cate resources.
The present report is the first survey of antiretroviral drug resistance in a small Brazilian municipality. Like all similar studies in the field, it should be interpreted with the understanding that resistance testing is insensitive to minor drug-resistant variants and thus may fail to tect strains resistant to previously used drugs and to de-tect decayed resistant variants transmitted to untreated subjects.
Brazilian surveillance studies on the prevalence of re-sistance mutations in treatment-experienced patients have reported variable prevalence figures of reduced suscepti-bility to antiretroviral agents. Mutation M184V, for in-stance, associated with high-level resistance to the NRTI lamivudine, has been recently reported in 67.7, 64, and 18% of patients failing HAART in the states of Rio de Janeiro (Couto-Fernandez et al. 2005), São Paulo (Rodrigues et al. 2005), and the Federal District (Cerqueira et al. 2004), respectively. Prevalence figures of resistance mutations are to be interpreted taking into consideration current local prescribing patterns, as well as recommen-dations, eligibility and access to HIV resistance testing for patient management.
In the present Miracema cohort, we found an overall low prevalence of reduced susceptibility to antiretroviral agents among 27 treatment-exposed patients. We recorded 9 samples with reduced susceptibility against RT inhibi-tors and 3 against PI. The prevalence of thymidine-asso-ciated mutations (4 cases) and of the M184V mutation (4 cases) clearly reflects the frequency with which combina-tions that include thymidine analogues (zidovudine and stavudine) and lamivudine are prescribed. Both patients with NNRTI-associated mutations had been treated with the NNRTI nevirapine, not efavirenz. Rapid emergence of high-level resistance is known to be a potential drawback of nevirapine-including regimens due to the drug’s low genetic barrier. No case of dual NRTI-NNRTI was recorded. Mutation T215Y/F, known to confer resistance to zidovudine and stavudine, was also not observed.
Among samples from 27 drug-naïve subjects, none had resistance mutations. These results corroborate find-ings from regional (Dumans et al. 2002) and national (Brindeiro et al. 1999, 2003) surveillance studies that re-ported a low prevalence of primary resistance in Brazil. Since antiretroviral drugs are readily available in Miracema since the early 1990s, our results seem to argue against the concern that resource-poor settings should not imple-ment widespread accessibility to standard of care HAART
Patient S ex A g e CDC Y ear of Time on stage sample therapy
Drugs used on treatment
a RT b TR mutations R T b PR mutations Subtype M 5 4 F 28 C 3 2002 naïve — S None S D60E, L63P B M 5 5 M 38 C 2 2003 naïve — S None S L63P B M 5 6 F 32 C 2 2003 naïve — S None S L10I, L63P , I93L B M 5 7 F 25 A 2 2003 naïve — S None S L10V
, D60E, L63P
B M 5 8 M 48 A 1 2003 naïve — S None S D60E, L63P B M 6 1 F 26 A 2 2004 naïve — S None S L63P , V77I B M 6 2 M 6 3 C 3 2004 18 mo AZT , D4T
, 3TC, LOP
S T69S S L63P B M 6 3 M 3 1 C 3 2004 1 yr AZT
, 3TC, LOP
S None S D60E, L63P B M 6 4 F 2 6 C 3 2004 5 mo AZT , D4T
, 3TC, EFV
S None S None B M 6 5 M 33 A 2 2004 naïve — S None S None B M 6 6 F 4 6 C 2 2004 2 mo AZT
, 3TC, LOP
S None S L63A B M 6 7 F 27 C 3 2004 naïve — S None S L53A, A71T B M 6 8 M 27 C 3 2005 naïve — S None S L63P , V77I B M 6 9 M 43 C 3 2005 naïve — S None S V77I B M 7 0 M 22 A 2 2005 naïve — S None S L63P , V77I, I93L B a
: drugs used as of sampling time are displayed in boldface, italic type;
AZT
(zidovudine); DDI (didanosine); 3TC (lamivudine);
D4T
(stavudine); NVP
(nevirapine); EFV
(efavirenz); SAQ
(saquinavir); IND (indinavir); RIT
(ritonavir); NFV
(nelfinavir); LOP
(co-formulation of lopinavir and ritonavir);
b
: drug resistance beta test interpretation result for the R
T
and R
T
genes:
S (susceptible); R (reduced susceptibility);
c
:
patients who were not on antiretroviral therapy as of sampling but who had been submitted to vertical transmission prophylaxis
(VTP) with
AZT
, 3TC, and NVP
873 873 873 873 873 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(8), D ecem ber 2005
combinations due to the possibility of sub-optimal adher-ence leading to the emergadher-ence and spread of drug-resis-tant strains. Continued surveillance studies on the preva-lence of drug resistant strains among treatment naïve and experienced patients will be necessary in large urban cen-ters. However, as the Brazilian epidemic spreads towards the innermost parts of the country, additional investiga-tions will also be needed in these settings.
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